Induction of feline immunodeficiency virus-specific cytotoxic T cells in vivo with carrier-free synthetic peptide

Abstract

The role of cellular immunity in the establishment and progression of immunosuppressive lentivirus infection remains equivocal. To develop a model system with which these aspects of the host immune response can be studied experimentally, we examined the responses of cats to a hybrid peptide containing predicted Tand B-cell epitopes from the gag and env genes of feline immunodeficiency virus (FIV). Cats were immunized with an unmodified 17-residue peptide incorporating residues 196 to 208 (from gag capsid protein p24) and 395 to 398 (from env glycoprotein gp120) of the FIV Glasgow-8 strain by using Quil A as an adjuvant. Virus-specific lymphocytotoxicity was measured by chromium-51 release assays. The target cells were autologous or allogeneic skin fibroblasts either infected with recombinant FIV gag vaccinia virus or pulsed with FIV peptides. Effector cells were either fresh peripheral blood mononuclear cells or T-cell lines stimulated with FIV peptides in vitro. Cytotoxic effector cells from immunized cats lysed autologous, but not allogeneic, target cells when they were either infected with recombinant FIV gag vaccinia virus or pulsed with synthetic peptides comprising residues 196 to 205 or 200 to 208 plus 395. Depletion of CD8+ T cells, but not CD4+ T cells, from the effector cell population abrogated the lymphocytotoxicity. Immunized cats developed an antibody response to the 17-residue peptide immunogen and to recombinant p24. However, no antibodies which recognized smaller constituent peptides could be detected. This response correlated with peptide-induced T-cell proliferation in vitro. This study demonstrates that cytotoxic T lymphocytes specific for FIV can be induced following immunization with an unmodified short synthetic peptide and defines a system in which the protective or pathological role of such responses can be examined.

Feline immunodeficiency virus vaccination: characterization of the immune correlates of protection

“…Microtiter plates (high binding; Greiner Laboritechnik, Dursley, Gloucestershire, United Kingdom) were coated overnight with either the synthetic peptide RAISSWKQRNRWEWRPD, representing an immunodominant linear neutralization site in the third variable (V3) region of FIV gp120 (10,24), or with an immunodominant epitope in the transmembrane glycoprotein (TM) (2,41) represented by the synthetic peptide CNQNQFFCK. Antibodies recognizing these FIV peptides were detected as described previously (14).…”

“…Virus-specific CTL were assayed in the peripheral blood, spleens, and lymph nodes of vaccinated and control cats. Effector cells derived from fresh or in vitro-restimulated lymphocytes were assayed for CTL activity against autologous or allogeneic skin fibroblast target cells derived from biopsy material collected prior to vaccination (14). Unstimulated, fresh cells were thawed, washed, and cultured for 18 to 24 h in complete RPMI 1640 medium supplemented with 100 IU of human recombinant interleukin-2 (a kind gift from Cetus, Emeryville, Calif.) per ml before use.…”

“…Effector cells with a viability of less than 99% (as assessed by trypan blue exclusion) were not used. Skin fibroblast target cells were labelled with 100 Ci of sodium 51 chromate (Amersham International plc., Amersham, United Kingdom) per/10 6 cells for 18 h at 37ЊC, washed three times, and then infected at 5 to 10 PFU per cell with recombinant vaccinia virus expressing either the gag (14) or env (43) gene product of FIV/GL-14 or FIV/PET, respectively, or with wild-type vaccinia virus for 1 h at 37ЊC. Unbound virus was washed away, and the cells were incubated for a further 2 h to allow optimal expression of the FIV Gag and Env products (3).…”

Three-color flow cytometry detection of virus-specific CD4+ and CD8+ T cells in the cat

“…One particular hurdle in the development of T cell assays for cats is that these animals constitute an outbred population for which MHC-typing has not yet been developed and for which virus-permissive, MHC-matched target cells are not readily available. Previously, both primary and SV40-immortalized autologous skin fibroblasts have been successfully used in 51 Cr-release CTL assays for feline leukemia virus and feline immunodeficiency virus (Flynn et al, 1994(Flynn et al, , 2000Köksoy et al, 2001). Hence, the use of immortalized autologous fibroblasts as APCs for T cell stimulation seemed an obvious choice.…”

“…So far, the tools to monitor feline T cell responses are restricted to the 'classical assays'. T lymphocyte proliferation tests for the cat have been described by Flynn et al (1994), but these do not distinguish between T cell subsets. Furthermore, cytotoxicity assays have been established to study T cell responses in cats infected with feline immunodeficiency virus and feline leukemiavirus (Tompkins and Tompkins, 1985;Song et al, 1992;Flynn et al, 1994Flynn et al, , 2000, but only provide semi-quantitative information about cytotoxic CD8+ T cells and do not permit a direct ex vivo analysis of antiviral T cell memory.…”